The basic aim of this proposed research is to obtain an understanding of the functional role of alpha-glycerol phosphate dehydrogenase (alpha GPDH) in cellular metabolism. Alpha GPDH catalyzes a reaction which acts as a branch point in mammalian metabolism. This fact along with the observations that there are multiple isozymic and tissue-specific forms of the enzyme, strongly suggest an important regulatory role. The elucidation of this regulatory role is the primary thrust of the proposed research. The current research project involves three complementary but distinct studies of the role of alpha GPDH in metabolism. a. Isolation and characterization of liver alpha GPDH. Studies will be continued on improving the newly developed isolation procedure. Kinetic analysis of the enzyme will be used to probe the interaction of the enzyme with its substrates with special emphasis being given to the binding of NADH and its expected large role in subunit interactions. The effect of cellular metabolites on the enzyme will be studied with the specific aim of elucidating any potential physiological regulatory mechanisms. b. Subunit interactions in skeletal muscle enzyme. Kinetic studies of the rabbit muscle enzyme will be initiated using the stopped-flow spectrophotometer. The enzyme will be examined for the now widely observed phenomenon of "half-of-the sites" reactivity. This phenomenon has been observed in other dehydrogenases and is a consequence of strong subunit interactions. Equilibrium binding studies of NADH interaction with the enzyme will be carried out in Conjunction with the kinetic studies and will be used to provide complementary information. c. Studies of heart muscle alpha GPDH. The isolation procedure used for the liver enzyme will be applied to the heart muscel enzyme with the hope of obtaining a purified preparation and determining the pattern of isozymes in this tissue. Preliminary steady-state kinetic studies will be initiated once the purified enzyme is obtained.